User Bulletin No. 61
Model 380A, 380B, 381A, 391, 392, 394 DNA Synthesizers
May 1991 (updated 06/2002)
SUBJECT:
Columns
40-nanomole Polystyrene: New Highly Efficient DNA Synthesis
New, smaller scale DNA synthesis columns are now available from Applied Biosystems. The 40-nanomole columns feature a novel polystyrene support that provides
efficient and economical synthesis for oligonucleotides of all lengths. Phosphoramidites can be used with these columns at a double dilution of 0.05 M, saving about 25%
of the cycle cost. In addition, the LOW cycle of the Model 391 and the primer cycle of
the 392 and 394 can be used with the 40-nanomole columns while maintaining excellent synthesis performance. Finally, these columns extend the life of phosphoramidites
in acetonitrile on the instrument to three weeks from the previous standard of two
weeks.
The 40-nanomole columns can be used on all Applied Biosystems DNA synthesizers,
and no new cycles are needed. Oligonucleotides of any length can be synthesized on
the new support. Currently, 40-nanomole columns are available only with standard
protecting groups, and therefore are not compatible with FOD™ (fast oligonucleotide
deprotection) reagents, except for those sequences that have a 3' T.
A New Support Material
A form of polystyrene has been developed as the solid support for these columns.
Manufactured as rigid beads that do not swell, this solid support has the attractive features of mechanical stability, rapid reaction kinetics, and the ability to be washed
quickly and efficiently with organic solvents. Parameters such as pore size, particle
size, and both polymerization formula and conditions have been optimized.1
Phosphoramidite DNA synthesis using controlled pore glass (CPO) supports can
attain 98-99% repetitive trityl yields. However, as oligonucleotide synthesis scale and
phosphoramidite excess are both reduced to achieve more economical cycles, the
deficiencies of CPG become more apparent. Although the silanol matrix of CPG is
exhaustively capped prior to synthesis, a small number of trimethylsilyl capping
groups are removed during detritylation with trichloroacetic acid, allowing the phosphoramidite to react with the nucleophilic silanols.2 Extraneous oligonucleotides can
be initiated and propagated during each synthesis cycle, resulting in 3' phosphate terminus failure sequences. These failures become more problematic at smaller scales.3
With an inert polystyrene support, side reactions during the preparation of the support
and during oligonucleotide synthesis are minimized by the lack of reactive functionality.
A 21-mer oligonucleotide was synthesized on a Model 380B DNA Synthesizer primer
equipped with both 40-nanomole polystyrene and 0.2-µmole CPG columns. Four milligrams of phosphoramidite were delivered each cycle to test the supports stringently,
using the fast cycle and 0.05 M phosphoramidites. Both the crude oligonucleotides
and those purified by the oligonucleotide purification cartridge (OPC™)4 were analyzed on the Model 270A by MICRO-GEL™100 Capillary Electrophoresis (CE)5 using
a new gel-filled matrix optimized for oligonucleotide analysis. The trityl yield for polystyrene was excellent (99.5%), but the CPG trityl yield was only fair (97.1%). The trityl
data was corroborated by the quantitation of product on CE. Examination of the CE
electropherograms suggests that undesired growth off the support may contribute to
the comparatively high (n-1 mer) contamination seen in the CPG samples. Trityl-selective OPC purification and subsequent CE analysis demonstrate that these contaminants are trityl bearing.
AB Model 270A
capillary electrophoresis
trityl:
polystyrene
CPG
99.5%
97.1%
CE product purity: 71%
54%
Figure 1. CEs of crude 21-mers.
Electropherograms of crude and OPC-purified samples also demonstrate the superior
quality of oligonucleotides synthesized on polystyrene supports. Syntheses conducted
with aged, partially degraded reagents also give better results when polystyrene supports are used. In addition to decreased extraneous propagation off the support, polystyrene supports offer coupling efficiency superior to that attainable with CPG. The
advantage is especially evident under impaired or more stringent synthesis conditions,
such as low phosphoramidite excess. The difference may reflect a more anhydrous
environment of the hydrophobic polystyrene.
Recommendations for Use
Dilution of The 40-nanomole polystyrene is especially intended for use with 0.05 M phosphoraPhosphoramidites midites to reduce cycle costs and still provide high-quality oligonucleotides. Both the
250- and 500-mg phosphoramidite bottles can be diluted with twice the normal volume
of acetonitrile to achieve a concentration of 0.05 M, as shown below:
Page 2 of 8
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
Table 1. 0.05 M Phosphoramidite Dilution Volumes
Phosphoramidite
Bottle Size
Acetonitrile Volume
Abz
500 mg
Abz
250
5.6
Gib
500
11.6
ib
250
5.8
Cbz
500
11.8
Cbz
250
5.9
T
500
13.2
T
250
6.6
G
11.2 mL
The auto dilute feature of the Model 392 and the 394 can provide 0.05 M phosphoramidites by using 500-mg bottles with the 1.0-g dilution option, or 250-mg bottles with
the 0.5-g dilution option.
Increased Phosphoramidite Lifetime
The 40-nanomole polystyrene columns contribute to efficient synthesis even with
0.05 M phosphoramidites that have been on the instrument for 21 days. A parallel
synthesis using a 0.2-µmole (small-scale) CPG column demonstrated inferior quality
after 14 days. The 40-nanomole polystyrene columns require fewer equivalents of
phosphoramidite for efficient synthesis.
AB Model 270A
capillary electrophoresis
CPG
trityl:
91.4%
CE purity:
50%
polystyrene
99.7%
91%
Figure 2. 21-day-old, 0.05 M phosphoramidites, concurrent syntheses of an 18-mer.
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
Page 3 of 8
Cycle Recommendations
These new columns may be used on all Applied Biosystems DNA synthesizers. No
new cycles are necessary.
Table 2. Cycle Recommendations for 40-nanomole Synthesis
Synthesizer
Cycle
380A, 380B
381
391
392-394
small-scale cycle (e.g., ssceaf 3, Version 2.01, 380B)
.2 UM cycle
LOW or .2 UM cycle
PRIMER or .2 UM CE
•
The 40-nanomole columns will support synthesis of oligonucleotides to 150 bases
on the above cycles with 0.05 M phosphoramidites.
•
The LOW cycle on the Model 391 is not recommended for synthesis of
oligonucleotides greater than 50 bases with 0.05 M phosphoramidites and the
40-nanomole columns.
CPG
PS
product peak
4.0%
product peak
10.7%
Figure 3. Comparison of crude 120-mers on CE. Syntheses conducted on a 391,
with .2 UM cycle, and 0.05 M phosphoramidites.
Page 4 of 8
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
Trityl Yields
Repetitive trityl yields are consistently at least 98-99% when used as described
above. Because of the small amount of trityl released each cycle, an accurate reading
of the trityls produced is difficult. Users who wish to read trityls may find it more convenient to dilute trityl solutions to 5.0 mL rather than 10.0 mL. The trityl concentrations
will be more appropriate, and less 0.1 M p-toluenesulfonic acid in acetonitrile will be
consumed.
Monitoring the trityl cation can be important, but the results must be interpreted with
caution. The trityl assay is only an indirect measure of synthesis efficiency. Certainly,
trityl cation yields of 98-99% must be present for a good synthesis. However, high trityl
yields can be present even when a poor synthesis occurs because this chemistry,
although highly refined, is not perfect. Unwanted side reactions do occur, some of
which contribute to the trityl cation released each cycle. Sites other than the 5'
hydroxyl group can participate in coupling (e.g., branched sites on the nucleic bases).
Low trityl yields (≤ 97%) always predict a less than optimal synthesis. In practical
terms, trityl yields of less than 95% do not allow successful synthesis of even short oligonucleotides. For the above reasons, the trityl assay must be viewed only as a preliminary yet convenient monitor of the synthesizer’s performance. The assay is useful
for the early detection and diagnosis of instrument-related problems. Many laboratories monitor their trityl fractions only by visual inspection. With experience, a failed
synthesis is detected this way. Evaluation of the oligonucleotide by PAGE, HPLC or
CE is much more informative than the trityl assay. Synthesizer or reagent problems
can be adequately diagnosed only by these methods, which are direct analyses of the
product.
Yield
Most users will find that the 40-nanomole scale provides sufficient quantities of oligonucleotide for their experiments. This scale is compatible with a single OPC purification. With 40-nanomole polystyrene, a typical primer gives 5-10 crude ODU
(150-300 µg) and 1-2 OPC-purified ODU (30-60 µg).
Protecting Groups
The current 40-nanomole columns (PN 401072-401075) have the standard protecting
groups Abz, Gib, and Cbz. Because T has no protecting group, T columns are also
compatible with FOD phosphoramidites.
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
Page 5 of 8
Cleavage and Deprotection
Covering the ammonia collection vials that are used in 40-nanomole syntheses with
Teflon-lined caps is especially important. Ammonia will leach a contaminant from
Wheaton rubber-lined caps (catalogue no. 225002, for 4-mL vials) into the DNA solution. This contaminant absorbs at 260 nm and 310 nm and will be seen as a fast moving band in a UV shadow gel of the product. The ratio of contaminant to DNA is such
that it might not be seen in larger scale syntheses but is readily apparent at the
40-nanomole scale. Teflon-lined caps may be obtained from Baxter (catalogue no.
B7508-1). For more details, see User Bulletin No. 32 (July 1986).
A very small amount of pyridine can be retained in the column from the oxidation step,
only to be washed into the collection vial along with the DNA during cleavage. This is
not noticeable at larger synthesis scales, but at the 40-nanomole scale it may be
detected in a UV spectrum of the diluted ammonium hydroxide by the spikes of the
pyridine absorbance superimposed on the normal DNA profile. The pyridine should be
of no concern because it is easily removable either by evaporation or by OPC. Users
who are still concerned may incorporate a 60-second acetonitrile wash at the beginning of the end procedure (before the 60-second reverse flush) to remove the pyridine
before cleavage starts. The deprotection time in ammonia for oligonucleotides made
on 40-nanomole polystyrene columns with standard protecting groups remains at
8-24 hours at 55° C.
Pyridine
200
350
No Pyridine
200
350
Figure 4. DNA spectra with and without pyridine
Manual Cleavage
Manual cleavage should be performed in the column with a syringe, rather than by
emptying the support into a vial containing concentrated ammonium hydroxide. A
quick acetonitrile, methanol or ethanol wash and subsequent 60-second reverse flush
is recommended before manual or auto cleavage of columns that have dried out since
synthesis was performed.
Page 6 of 8
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
References
1
Andrus, A. and McCollum, C. “A New Support for Automated Oligonucleotide
Synthesis,” presented at the Symposium on Solid Phase Synthesis, Oxford, U.K.,
August 1989.
2
Port, R.T., Usman, N. and Ogilvie, K.K. (1988) “Derivatization of Controlled Pore
Glass Beads for Solid Phase Oligonucleotide Synthesis,”
Biotechniques 6:768-775.
3
Andrus, A. and McCollum, C. “A New Support for Automated Oligonucleotide
Synthesis,” presented at the Ninth International Round Table: Nucleosides,
Nucleotides and Their Biological Applications, Uppsala, Sweden, July 1990.
4
McBride, L.L., McCollum, C., Davidson, S., Efcavitch, J.W., Andrus, A. and
Lombardi, S. (1988) “Oligonucleotide Purification Cartridge,”
Biotechniques 6:362-367.
5
Moring, S.E., Colburn, J.C., Grossman, P.D. and Lauer, H.H. (1990) “Analytical
Aspects of an Automated Capillary Electrophoresis System,” LC-GC 8:34.
User Bulletin No. 61: 40-nanomole Polystyrene: New Highly Efficient DNA Synthesis Columns
Page 7 of 8
© Copyright 2002 Applied Biosystems. All rights reserved.
These columns contain microporous particles and are required to be identified by the following legend:
MADE IN THE UK
This product contains media manufactured under one or more of US patents 4,224,415; 4,256,840; 4,382,124; and 4,501,826 and the
purchaser is entitled to utilize this product under US Patent 4,297,220 in analytical, preparative and process chromatography (excluding
treatment of fruit juice), medical diagnostics and catalyst and chemical synthesis supports and for no other use.
Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
All other trademarks are the sole property of their respective owners.
Stock No. 108UB12-01